MyoD forms micelles which can dissociate to form heterodimers with E 47 : Implications of micellization on function ( analytical ultracentrifugation / helix - loop - helix proteins / transcriptional regulation )

MyoD is a member of a family of DNAbinding transcription factors that contain a helix-loop-helix (HLH) region involved in protein-protein interactions. In addition to self-association and DNA binding, MyoD associates with a number of other HLH-containing proteins, thereby modulating the strength and specificity of its DNA binding. Here, we examine the interactions of full-length MyoD with itself and with an HLH-containing peptide portion of an E2A gene product, E47-96. Analytical ultracentrifugation reveals that MyoD forms micelles that contain more than 100 monomers and are asymmetric and stable up to 36°C. The critical micelle concentration increases slightly with temperature, but micelle size is unaffected. The micelles are in reversible equilibrium with monomer. Addition of E47-96 results in the stoichiometric formation of stable MyoD-E47-96 heterodimers and the depletion of micelles. Micelle formation effectively holds the concentration of free MyoD constant and equal to the critical micelle concentration. In the presence of micelles, the extent of all interactions involving free MyoD is independent of the total MyoD concentration and independent of one another. For DNA binding, the apparent relative specificity for different sites can be affected. In general, heterodimer-associated activities will depend on the selfassociation behavior of the partner protein. Implicit in all current models of gene regulation are thermodynamic linkages between protein-protein association and protein-DNA binding. Proteins containing the basic helixloop-helix (bHLH) motif require oligomerization to form either homomeric or heteromeric complexes with high-affinity DNA-binding activity (1-3). These proteins regulate gene expression by binding to DNA containing the consensus E-box sequence CANNTG (2, 4, 5). The HLH region is responsible for protein dimerization, and the basic region is directly involved in DNA binding (3, 6-10). MyoD is a transcriptional activator that plays a principal role in muscle differentiation (11, 12). It is one of a family of bHLH-containing muscle determination/differentiation factors that appear to function in vivo as heteromers with E2A gene products E12 and E47 (13, 14). A 68-residue peptide containing the bHLH region of MyoD is sufficient for conversion of fibroblasts to myoblasts (15). The bHLH region of MyoD (MyoD-bHLH) forms dimers and tetramers in the absence of DNA (16) and binds DNA as a dimer (10). The oligomeric states of full-length myogenin, MyoD, and E12 have been assessed by using thin-zone sucrose-gradient sedimentation (17). These studies demonstrate that in the absence of DNA both myogenin and MyoD reversibly form oligomers larger than dimer size and that E12 predominantly The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. forms dimers. However, the reequilibration between oligomeric forms during the course of these experiments made it difficult to ascertain the stoichiometries. To obtain direct evidence about the association behavior of MyoD? analytical ultracentrifugation experiments have been carried out on full-length MyoD, both alone and mixed with an HLH-containing peptide of E47 (E47-96). MyoD exhibits micellular behavior-i.e., highly cooperative and reversible selfassociation-forming asymmetric assemblies containing more than 100 monomers. Such behavior-could have significant implications for the function of MyoD. MATERIALS AND METHODS MyoD Preparation. Full-length MyoD and MyoD-bHLH were expressed and purified as described (16, 18). The protein (1 mg) was size fractionated on a Superose 6 column by using a Pharmacia fast protein liquid chromatography (FPLC) system. TIhe column running buffer was 10% (vol/vol) glycerol/20 mM Hepes, pH 7.6/2 mM EDTA/p.1% Triton X-100/0.1 M NaCl/1 mM dithiothreitol/1 mM phenylmethylsulfonyl fluoride/2 ,ug each of pepstatin and leupeptin per ml. Fractions (0.2 ml) were collected, and aliquots were analyzed by a gel mobilityshift assay. Electrophoretic mobility-shift assays were performed essentially as described (18-20) by using 5% miniacrylamide gels. Purified protein was incubated for 5 min at room temperature in 20mM Hepes, pH 7.6/50mM KCl/3 mM MgCl2/1 mM EDTA/ 0.5% Nonidet P-40 with 500 ng of an oligonucleotide probe containing the muscle creatine kinase (MCK) enhancer-binding sites for MyoD. E47-96 Construction, Expression, and Purification. A phage T7 RNA polymerase-driven expression plasmid (21) encoding the fragment of E47 containing the bHLH region (residues 317-413, numbered as described in ref. 1) was constructed in the pRSET A vector (Invitrogen). BL21(DE3) cells containing the pLysS plasmid (22) were transformed with this plasmid to produce E47-96. The resultant protein included an Ala-Arg dipeptide at the N terminus and a Met at the C terminus. Cells were grown at 37°C on minimal medium to an OD650 of 0.8, then induced with 1 mM isopropyl P3-D-thiogalactoside and harvested 4 h later. Protein purification was essentially as described for MyoD-bHLH (16), except lower salt concentrations were used for lysis and cation-exchange chromatography. Cells were lysed by cycles of freezing and thawing in the presence of 50 mM Hepes, pH 7.6/1 mM EDTA/1 mM MgCl2, 0.1% Triton X-100/5 mM dithiothreitol/1 mM phenylmethylsulfonyl fluoride. Crude cell extract was loaded onto an SP Sepharose column Abbreviations: HLH, helix-loop-helix; bHLH, basic HLH; cmc, critical micelle concentration; MyoD-bHLH, bHLH region of MyoD. ITo whom reprint requests should be addressed. lPresent address: Department of Protein Engineering, Genentech, Inc., South San Francisco, CA 94080. ttDeceased, March 28, 1995.